ANALYSIS OF LSAMP GENE AS A TUMOR SUPPRESSOR IN NEUROBLASTOMA A THESIS SUBMITTED TO THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS AND THE INSTITUTE OF ENGINEERING AND SCIENCE OF BILKENT UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE BY ATIL ÇAĞDAŞ SAYDERE DECEMBER 2009
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ANALYSIS OF LSAMP GENE AS A TUMOR SUPPRESSOR IN
NEUROBLASTOMA
A THESIS SUBMITTED TO
THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
AND THE INSTITUTE OF ENGINEERING AND SCIENCE OF
BILKENT UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
MASTER OF SCIENCE
BY
ATIL ÇAĞDAŞ SAYDERE
DECEMBER 2009
i
SIGNATURE PAGE
I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Master of Science.
____________________________
Assist. Prof. M. Cengiz Yakıcıer
I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Master of Science.
____________________________
Assist. Prof. Özlen Konu
I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Master of Science.
____________________________
Assoc. Prof. Hilal Özdağ
Approved for the Institute of Engineering and Science
____________________________
Prof. Dr. Mehmet Baray
Director of Institute of Engineering and Science
ii
ABSTRACT
ANALYSIS OF LSAMP GENE AS A TUMOR SUPPRESSOR IN
NEUROBLASTOMA
ATIL ÇAĞDAŞ SAYDERE
M.Sc. in Molecular Biology and Genetics
Supervisor: Assist. Prof. M. Cengiz Yakıcıer
December 2009, 59 pages
Neuroblastoma constitutes approximately 10 % of all childhood tumors with a
worldwide considerable morbidity and mortality. Frequently, in children under the
age of 1, it can spontaneously regress and transform into a benign tumor. However,
in children older than age of 1 the disease often behaves aggressively and
metastasizes to other organs. This unpredictable behavior of unknown origin makes
therapeutic applications ineffective.
Limbic system associated membrane protein gene (LSAMP) functions in neurite
growth, axonal guidance and acts as a cell adhesion and recognition molecule.
Recent studies revealed its association to several cancer types and proposed a
potential tumor suppressor role. Markers in the LSAMP gene region were also shown
to be homozygously deleted in neuroblastoma. In the framework of this study, we
investigated LSAMP gene in respect of its potential tumor suppressor role in
neuroblastoma. 6 clinical patient samples and 2 neuroblastoma cell lines were
studied via PCR methodology to detect any loss in LSAMP gene.
Immunohistochemistry (IHC) was applied to 6 neuroblastoma tissue sections to
determine protein level changes of LSAMP. Moreover, expression analysis in a set of
brain tumors was performed. As a result of these efforts, one possible LOH and one
homozygous deletion in two different patients were observed. Low levels of LSAMP
protein in all of the tumor samples compared to controls were recorded.
iii
Downregulation of LSAMP in brain tumors was detected. Based on these results,
LSAMP is suggested as a candidate tumor suppressor in neuroblastoma and in
broader aspect for nervous system tumors.
iv
ÖZET
LSAMP GENĐNĐN NÖROBLASTOMALARDA TÜMÖR BASKILAYICI
OLARAK ANAL ĐZĐ
ATIL ÇAĞDAŞ SAYDERE
Moleküler Biyoloji ve Genetik Bölümü Yüksek Lisansı
Danışman: Assist. Prof. M. Cengiz Yakıcıer
Aralık 2009, 59 sayfa
Nöroblastoma dünya çapında önemli morbidite ve mortaliteye sahip olup tüm
çocukluk çağı tümörlerinin yaklaşık %10’unu oluşturmaktadır. Sıklıkla, 1 yaş
altındaki çocuklarda, kendiliğinden gerileyerek ve iyi huylu bir tümör haline
dönüşmektedir. Ancak, 1 yaş üzeri çocuklarda genellikle agresif davranır ve diğer
organlara metastaz olur. Kaynağı bilinmeyen bu beklenmedik davranış tedavi
uygulamalarını etkisiz hale getirir.
Kromozom bölgesi 3q13.3 üzerinde bulunan limbik sistem ilişkili membran proteini
geni (LSAMP) sinir hücrelerinin büyümesinde ve akson rehberliğinde görevlidir.
Ayrıca, bir adezyon ve hücreler arası tanıma molekülü gibi davranır. Son yıllarda
yapılan çalışmalarda çeşitli kanser türleriyle ilişkisi gösterilmiş ve potansiyel bir
tümör baskılayıcı rolü önerilmiştir.Ayrıca LSAMP geni bölgesindeki markörlerin
homozigot kayıplara uğradığı gösterilmiştir. Bu çalışma çerçevesinde, LSAMP
geninin nöroblastomada olası bir tümör baskılayıcı rolü araştırıldı. 6 klinik hasta
numunesi ve 2 nöroblastoma hücre hattı PCR yöntemi ile LSAMP gen kaybını ortaya
çıkarmak için çalışıldı. 6 nöroblastoma doku kesitinde LSAMP protein düzeyindeki
değişiklikleri belirlemek için immunohistokimya (ĐHK) uygulandı. Bir grup beyin
tümöründe ifade analizi yapıldı. Đki farklı hastada bu çabalar, olası bir LOH ve bir
homozigot kayıp sonuçları olarak gözlemlendi. Kontrol grubuna göre tüm tümör
örneklerinde LSAMP proteinin düşük seviyeleri kaydedildi. Beyin tümörlerinde
Table 11: Reaction setup in quantitative RT-PCR experiments ................................ 29
Table 12: The relative expression levels of LSAMP and GAPDH in different types
and grades of brain tumors revealed by semi quantitative PCR ................................ 41
Table 13: Quantitative RT-PCR results of LSAMP in brain tumors ......................... 42
Table 14: Multiplex-PCR analysis of LSAMP in neuroblastoma tissues showing
possible deletions ....................................................................................................... 45
xiii
ABBREVIATIONS
ACTB: B-actin
bp: Base Pair
cDNA : Complementary DNA
CGH: Comparative Genomic Hybridization
∆H2O: Double Distilled Water
DM: Double Minutes
DNA: Deoxyribonucleicacid
dNTP: Deoxyribonucleotide Triphosphate
EtBr: Ethidium Bromide
EDTA: Ethylenediaminetetraacetic acid
FBS: Fetal Bovine Serum
FISH: Fluorescent in situ Hybridization
g: Gram
GAPDH: Glyceraldehyde-3-Phosphate Dehydrogenase
HCC: Hepatocellular carcinoma
HSR: Homogenously Staining Regions
IHC: Immunohistochemistry
LOH: Loss of Heterozygosity
LSAMP: Limbic System Associated Membrane Protein
min: Minute
ml: Mililiter
mM: Milimolar
MP-PCR: Multiplex PCR
mRNA: Messenger RNA
MYCN: v-myc Avian Myelocytomatosis Viral Related Oncogene,
Neuroblastoma Derived
NaCl: Sodium Chloride
PBS: Phosphate Buffered Saline
PCR: Polymerase Chain Reaction
xiv
pm: Picomole
Q-RT-PCR: Quantitative Real Time PCR
RNA: Ribo Nucleic Acid
Rpm: Revolutions Per Minute
Sec : Second
TAE: Tris Acetate EDTA Buffer
Tris: Tris (Hydroxymethyl)-Methylamine
UV: Ultraviolet
w/v: Weight/Volume
µg: Microgram
µl: Microliter
µm: Micrometer
1
1 INTRODUCTION
Neuroblastoma was first identified and characterized as a tumor by Virchow in 1864
and by Marchand in 1891, respectively (Hayes PA, 1989; Voute PA, 1984). Since
their identification, the tumor has succeeded to stimulate interests of the researchers
by its distinct characteristics. The neuroblastoma tumor tissues either regress
spontaneously as observed especially in infants or they mature into benign tumors
called ganglioneuroma (Brodeur and Castleberry, 1993). However, it is almost
impossible to predict the behavior and most children over 1 year of age already have
tumor metastasized into other organs at the time of diagnosis (Brodeur, 2003). The
heterogeneous behavior of the disease prevented comprehensive clinical studies and
resulted in poor prognosis until studying the tumor with the tools of molecular
genetics and biochemistry.
1.1 Epidemiology
Neuroblastoma is the most common extracranial solid tumor seen in pediatric
population. It accounts for 8-10 % among all childhood cancers and it is the most
common diagnosed cancer type for infants (Gurney et al., 1997). Neuroblastoma is
diagnosed in United States and Canada one case in 7000 live births per year and
approximately 700 new cases per year are observed (Brodeur and Castleberry, 1993).
In Europe compared to USA and Canada more than two times higher, 1500 cases
occur per year (Gao et al., 1997; Spix et al., 2006). Among all cancers diagnosed in
European and USA infants, neuroblastoma accounts for about 28% (Gurney et al.,
1997; Spix et al., 2006). Almost the same incidence rate for the rest of the world
according to reported cases are observed (Brodeur and Castleberry, 1993). The
incidence has a peak before the age 1 and the median age of the patients diagnosed
with neuroblastoma is 18 months (Brodeur and Castleberry, 1993; Heck et al., 2009).
2
There is an equivalent or slightly higher prevalence of the disease in boys than girls
in most countries. There is no difference between the ethnic groups for the incidence
of the disease (Gao et al., 1997; Ries et al., 2005; Spix et al., 2006).
1.2 Pathogenesis and Aetiology
1.2.1 Pathogenesis
Neuroblastoma is a malignant tumor originating from the neural crest cells which
arise in the third to fourth week of embryonic development. Some pluripotent cells or
neuroblasts of this layer can form the components of the sympathetic nervous system
by differentiating into sub populations (Fig.1). These cells invaginate and migrate
along the neuraxis and accumulate in sympathetic ganglia, adrenal medulla and
different sites of the bone and soft tissue (Gray H, 2005). The distribution of these
cells into the sub regions of the sympathetic system correlates with the primary
diagnosis sites of the neuroblastoma. In most cases, approximately 40%, the origin
side of the tumor is one of the adrenal glands or in other cases it is observed in the
chest, abdomen and pelvic (Gao et al., 1997; Ries et al., 2005).
Among other known human malignancies, neuroblastoma distinguishes itself by an
enigmatic behavior in which some cells undergo a spontaneous regression from a
malign state to a benign state while others persistently progress (Brodeur and Maris,
2006) . The tumor may undergo differentiation and/or apoptosis in patients under 1
year of age in contrast with patients older than 1 year of age where it shows an
aggressive behavior and leads to death (Brodeur, 2003; Nakagawara, 1998).
3
Figure 1: The lineages derived from neural crest and the origin of neuroblastoma. (Nakagawara and Ohira, 2004)
In order to facilitate diagnosis and prognosis of the disease a classification system
called Shimada System which classify patients into three risk groups; low,
intermediate and high was created in 1984 (Shimada et al., 1984). It was used to
determine the curability of the patient ; low risk and intermediate risk patients which
usually comprise infants have a higher chance of cure compared to high risk group
patients who have poor diagnosis and outcome (Maris, 2005). The majority of the
patients older than 18 months have the disease metastasized to lymph nodes, liver,
bone and bone marrow but despite the intensive therapies followed by autologous
bone marrow transplant, more than half of the patients cannot survive (Spix et al.,
2006)
The recent advances allowed the disease to be re-classified into different stages and
risk groups by using factors like differentiation state, MYCN amplification, age,
Schwannian stroma content and mitosis-karyorrhexis index at the time of diagnosis
(Maris et al., 2007; Shimada et al., 1999). The efforts of International Neuroblastoma
Risk Group (INRG) developed a classification system in modifying the previously
used Schimada System. According to this classification schema the tumors are
4
categorized into three risk groups; low, intermediate and high and different stages of
the disease is defined from 1 to 4 where 4 represents the metastasized tumor (Maris
et al., 2007). One stage called 4s which is special for the patients younger than 1 year
of age and seen nearly 5% of the patients is also described. In this case, the tumor is
spread to liver, skin or bone marrow but show a spontaneous regression (D'Angio et
al., 1971).
1.2.2 Aetiology
The sporadic occurrence of the disease makes it challenging to study and there is not
much known about the aetiology of the disease but as a common reason in
tumorigenesis environmental effects are thought to play a role for the development of
the disease (Brodeur, 2003). Among these environmental factors, using medications
like pain killers, exposure to electro-magnetic fields, the use of sex hormones and
vitamins has been hypothesized for the aetiology of the disease (Carachi, 2002). In
recent studies maternal alcohol consumption, paternal exposure to nonvolatile and
volatile hydrocarbons, wood dusts and solders, use of diuretics and low birth weight
are suggested to be positively associated with the disease (Heck et al., 2009).
1.3 Genetic Aberrations in Neuroblastoma
The molecular pathology of neuroblastoma is still poorly understood. However,
common genetic alterations have been observed in patients diagnosed with
neuroblastoma and these alterations are used for risk stratification. The best
characterized genetic alterations include amplification of the proto-oncogene MYCN,
gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Based on these
aberrations, neuroblastoma is classified into three genetic sub groups. Beside MYCN
on chromosome arm 2p which was previously identified as an oncogene, further
efforts are focused on identifying any candidate tumor suppressor genes or
5
oncogenes located within the regions of loss or gain, respectively. Until now, no new
genes that drive neuroblastoma development and progression have been successfully
characterized.
1.3.1 Genomic Gains:
1.3.1.1 Ploidy:
DNA content is an important prognostic marker for the neuroblastoma patients.
Although in most cases tumors have diploid karyotypes, a distinct case for lower
stage neuroblastoma patients exist as they may often have hyperdiploid or near
triploid (Kaneko et al., 1987). Thus, during prognosis the karyotype analysis of
neuroblastomas is performed to determine the ploidy status and having near-triploidy
is more favorable for the patients associated with longer survival. However, it is
more significant for infants compared to children older than 1 year of age. The
reason behind is that less aggressive tumlors have a major defect in mitosis which
results in whole chromosomal gains and losses where aggressive tumors additionally
imperfect in genomic stability which ends in structural rearrangements (Brodeur,
2003; Look et al., 1984).
1.3.1.2 MYCN Genomic Amplification:
The well known associated marker of neuroblastoma is the genomic amplification of
MYCN which occurs in 20% of patients and strongly correlates with higher stages of
the disease and poor outcome (Brodeur and Seeger, 1986; Schwab et al., 2003;
Shimada et al., 1999). MYCN gene is located on chromosome 2p24 locus and it was
identified in neuroblastoma cell lines as an amplified DNA sequence homologous to
proto-oncogene c-MYC. The amplified region that contains MYCN chromosome
6
2p24 locus form double minute chromosomes (DM) and they are integrated linearly
to random chromosomes resulting in homogenously staining regions (HSR) that may
contain copies up to 500 (Schwab et al., 1983) (Fig. 2). Rather than a mutation in this
amplified region, it is suggested that overexpression of wild type MYCN leads to
tumorigenesis as overexpressing MYCN in neural crest of transgenic mice can
develop neuroblastomas and decreasing MYCN mRNA levels by using antisense
MYCN can induce differentiation of human neuroblastoma cell lines (Schmidt et al.,
1994; Weiss et al., 1997).
Figure 2: MYCN amplification pattern; A) Amplificat ion patterns of double minutes (DM) and homogenously staining regions (HSR) are observed in solid tumors (Albertson et al., 2003) B) FISH image of MYCN amplification presented in DM (Maris et al., 2007) C) HSR for MYCN amplification in neuroblastoma cell line NGP (Schwab et al., 2003)
1.3.1.3 Gain of 17q:
Extra copies of chromosome arm 17q are observed in more than half of the
neuroblastomas. The gain of 17q may occur independently but more often results
from unbalanced translocation events with chromosome 1p or 11q and associated
with poor outcome (Bown et al., 1999; Caron, 1995). The gene dosage effect of one
or more genes in extra copies is thought to provide a selective advantage to cells
resulting in aggressive phenotype (Lastowska et al., 2002). Although there is no
exact site of breakpoint, the region of 17q22-qter has been suggested as carrying
7
genes that promotes cell survival (Van Roy et al., 1997). The most of the genes have
not been identified yet but among the known genes overexpression of BIRC5
(survivin) which encodes an inhibitor of apoptosis proteins has been proposed for the
reason of aggressive phenotype seen in gain of 17q (Islam et al., 2000). In some
cases segmental gains of 1q, 5q and 18q are also observed but their characterizations
still remain in question (Schwab et al., 2003).
1.3.2 Genetic Deletions and Allelic Losses:
1.3.2.1 Loss of 1p:
The most common loss of heterozygosity (LOH) seen in neuroblastoma patients is
the loss of 1p which is seen about 30-35% of the cases (Brodeur, 2003). Deletions in
chromosome region 1p correlate with the advanced stages of the disease and
frequently associated with MYCN amplification and unbalanced translocation with
17q, t (1; 17) (Caron, 1995). Studies identified the smallest region that overlaps in
tumors with loss of 1p as the locus 1p36 and loss of putative tumor suppressors in
this region is thought to be responsible for disease progression (Caron et al., 2001).
However, there is no consensus about the role of this locus independent of other
factors as there are inconsistent data presented by different groups.
1.3.2.2 Loss of 11q:
Approximately 35% of the diagnosed neuroblastoma patients have deletions at
chromosome region 11q. Translocations involving 11q21 and 11q22, deletion at
11q23, inversion at 11q21-11q23 and allelic losses are observed (Brodeur and Maris,
2006). In most of the cases allelic losses are reported and it is negatively correlated
with MYCN amplification. Deletion in 11q is frequently found in neuroblastomas
8
without MYCN amplification (Guo et al., 1999; Plantaz et al., 2001). However, the
loss at this region associates with the advanced stages of the disease and poor
prognosis. The existence of tumor suppressor genes in this region is thought to be
responsible for malignant progression upon inactivation by allelic losses in MYCN
single copy neuroblastomas (van Noesel and Versteeg, 2004).
1.3.2.3 Losses at Other Chromosomes:
Beside chromosome regions 1p and 11q, allelic losses at chromosome arms 2q, 3p,
4p, 9p.12p, 14q, 15q, 16p and 19q are observed in lower frequencies. Several genes
are identified in these regions as tumor suppressors or important regulators of
cellular functions, but further investigation are needed for their prognostic
Table 4: List of brain tumors of different types and grades used in this study
3.1.4 Cell Culture:
3.1.4.1 Cell Lines:
Two frozen neuroblastoma cell lines SK-NAS and CLB-MA1 (LT) were obtained
from Dr. Valérie Combaret of Oncologie Génétique Centre Léon Bérard, France.
Cells were tested and mycoplasma free. Cell lines in cryotubes were immediately
stored in liquid nitrogen tanks after their arrival in dry ice.
3.1.4.2 Media and Solutions
Two cell lines SK-NAS and CLB-MA1 (LT) were cultured in 25 ml flasks
(Greigner-Bio) as monolayer. Cell lines were grown in RPMI-1640 (Biological
23
Industries) supplied with 10% FBS (Sigma), 50mg/ml penicillin / streptomycin and
non-essential amino acids (Biochrom AG). Cell lines were cultured at 37°C in an
incubator with 5% CO2 (Heto-Holten, Surrey, UK). Cells were handled in sterile
laminar hoods (Heto-Holten, Surrey, UK). Reagents were kept at 4°C except
Trypsin – EDTA which was stored at -20 °C and pre-heated to 37°C before use.
3.1.4.3 Antibody
Rabbit polyclonal antibody to LSAMP (ab64427) was purchased from Abcam
(Cambridge, USA).
3.2 Methods:
3.2.1 The cDNA Synthesis:
RevertAidTM First Strand cDNA synthesis kit (#k1622) (Fermentas, USA) was used
for all cDNA synthesis reactions. All reagents were provided by the kit. Total RNA
samples isolated from brain tumor patients were reverse transcribed according to
manufacturer’s protocol. 2 µg of RNA template was mixed with 1 µl oligodT and
DEPC treated water is added to 12 µl. Mixture was gently pipetted and incubated at
70°C for 5 minutes. After incubation 4 µl of 5X reaction buffer, 1 µl of RibolockTM
Ribonuclease inhibitor and 2 µl of dNTP mixture were added in order. The mixture
was gently pipetted and incubated at 37°C for 5 minutes. In next step, 1 µl of Revert
Aid TM M-MuLV Reverse Transcriptase is added and incubated at 42°C for 1 hour
proceeded by 10 minutes at 70°C. The products were stored at -20°C for further use.
24
3.2.2 Multiplex PCR Reaction:
Multiplex PCR (MP-PCR) was performed by using genomic primers for LSAMP and
GAPDH. All PCR reactions were carried out by using Techne-512 PCR machine
(Techne Inc). Optimal conditions were obtained by changing primer concentrations
and temperature. Reaction conditions and final concentrations of reagents used in
master mix for one tube (25 µl) are listed in Table 5 and Table 6, respectively.
Templates containing 100 ng or 200 ng of DNA were used in reactions. All resulting
PCR products were assessed by running in 2% agarose gels with EtBr and visualized
under UV.
Reagent Stock
Concentration
Final
Concentration
Master
Mix
Setup
Reaction Buffer 10 X 1 X 2.5 µl
LSAMPgnf54 10 pm 0.4 pm 1 µl
LSAMPex3R 10 pm 0.4 pm 1 µl
GAPDH_GENOMIC_20070206_F 10 pm 0.15 pm 0.375 µl
GAPDH_GENOMIC_20070206_R 10 pm 0.15 pm 0.375µl
MgCl2 50 mM 1.5 mM 0.75 µl
DyNAzymeTM II DNA Polymerase 2 U/µl 2 U 1 µl
∆dH2O - - Add to 25
µl*
Table 5: MP-PCR master mix reagents and concentrations
25
Reaction Steps Cycle Numbers
5 min. of initial denaturation at 95°C
1
30 sec. at 95°C
35 30 sec. at 60°C
35 sec. at 72°C
10 min. at 72°C 1
Table 6: MP-PCR Reaction conditions
3.2.3 Semi-Quantitative PCR Reaction:
Brain tumor cDNAs were tested for LSAMP expression level by semi Q-PCR
method. GAPDH was selected as a housekeeping gene before starting to experiments
and all samples were tested for GAPDH expression. All PCR reactions were done in
Techne-512 PCR equipment (Techne Inc). Optimal conditions were obtained by
changing primer concentrations and temperature. Reaction conditions and final
concentrations of reagents used in master mix for one tube (20 µl) are listed in Table
7 and Table 8, respectively. All resulting PCR products were assessed by running in
agarose gels with EtBr and visualized under UV.
26
Reagent Stock
Concentration
Final
Concentration
Master Mix
Setup
Reaction Buffer 5 X 1 X 4 µl
LSAMP Forward Primer# 10 pm 0.5 pm 1 µl
LSAMP Reverse Primer# 10 pm 0.5 pm 1 µl
PhireTM Hot Start DNA
polymerase - - 0.4 µl
∆dH2O - - Add to 20 µl*
Table 7: Semi-Q-PCR master mix reagents and concentrations
Reaction Steps Cycle Numbers
30sec. initial denaturation at 98°C
1
5 sec. at 98°C
30 5 sec. at 63°C
20 sec. at 72°C
1 min. at 72°C 1
Table 8: Semi-Q-PCR Reaction conditions
27
3.2.4 Quantitative RT-PCR Reaction:
SYBR Green I method recommended by manufacturer was applied for analysis of
LSAMP expression in brain tumors and normal brain tissue. LSAMP primer mix and
Beta-actin gene (ACTB) primers were purchased from OriGene and used in all
reactions. Beta-actin gene was used as internal control. 37 tumor and 3 normal brain
samples were tested in duplicates for both LSAMP and beta-actin gene (ACTB).
Reaction mixture setup for one tube and conditions are listed in Table 9, 10 and 11.
Corresponding readings of expression for both genes in tumors and normal samples
were used in calculating expression differences (Table 12). The relative expression
of LSAMP in tumors compared to normal brain tissues has been calculated by delta -
delta CT method. The formula used in these calculations is as below:
• Relative Expression Ratio =
[E (LSAMP) ∆Ct
LSAMP(Ct normal - Ct tumor) / E (ACTB)
∆CtACTB
(Ct normal - Ct tumor) ]
In this formula, E (LSAMP) and E (ACTB) represent the primer efficiencies of LSAMP and
ACTB primers, respectively. Efficiency of the primers was approximated as 100%
therefore a two fold increase at each cycle (E (LSAMP) = 2, E (ACTB) = 2). ACTB gene (B-
actin) was used as reference for normalization. Ct normal values correspond to Ct
values of normal brain samples, where Ct tumor belongs to Ct values of brain
tumors. Results were log2 transformed and plotted (Fig.11). Statistical analysis of
significance was performed by using one sample t-test at 95% confidence interval
(H0 = No difference exists in tumors compared to normal = 0).
28
Reaction Mixture Volume
2X Master mix (Bio-Rad IQ ) 12.5 µl
LSAMP primer mix 1 µl
Template cDNA 1 µl
dH2O 10.5 µl
Mineral oil 12.5 µl
Total 37.5 µl
Table 9: Q-RT-PCR reaction mixture for LSAMP gene
Reaction Mixture Volume
2X Master mix (Bio-Rad IQ ) 12.5 µl
Beta-actin forward primer (10pm) 1 µl
Beta-actin reverse primer (10pm) 1 µl
Template cDNA 1 µl
dH2O 9.5 µl
Mineral oil 12.5 µl
Total 37.5 µl
Table 10: Q-RT-PCR reaction mixture for beta-actin (ACTB) reference gene
29
Reaction Steps Cycle Number
5 min. of initial denaturation at 95°C 1
15 sec. at 95°C
40 15 sec. at 62°C
15 sec. at 72°C
10 min. at 72°C 1
Melting Curve Step
1 min. at 95 °C
1 30 sec. at 55 °C
30 sec. at 95 °C
Table 11: Reaction setup in quantitative RT-PCR experiments
3.2.5 Agarose Gel Electrophoresis
2% agarose gels with 30ng/µl EtBr were prepared to run PCR products. 2µl of 6X
DNA loading dye was added to 10µl of each PCR product and gently pipetted. 1X
TAE buffer was added into gel electrophoresis equipment and gel tray was placed
properly. Gene Ruler DNA Ladder Mix of Fermentas (100-10,000bp) (Burlington,
Canada) was used as size marker and loaded into the first well. Samples were loaded
into separate wells which are horizontally aligned and run vertically under 100 V for
approximately 30 minutes. Visualization and photography of DNAs were carried out
by using Bio-Rad (California, USA) Transilluminator equipment under UV light of
340 nm wavelength.
3.2.6 Thawing and Culturing Cell Lines
Cryotubes were removed from liquid nitrogen tanks and thawed at 37°C. Lids of the
tubes were carefully loosened and pressure inside the tubes was decreased. The cells
were resuspended gently by using a pipette and 5ml of growth medium pre-heated to
37°C was added into each tube inside the sterile hood. The cells were centrifuged at
30
1500 rpm for 5 minutes. Supernatants were discarded and the pellets were
resuspended in 5 ml growth medium to plate into 25 ml flasks. Flasks were stored in
a humidified incubator at 37°C with 5% CO2. Culture mediums were refreshed in
the next day.
3.2.7 Growth and Passaging of Cell Lines
Cells were examined under bright field microscope everyday for their confluency
and culture mediums were refreshed. All culture mediums and enzymes were pre-
heated to 37°C. Inside the sterile hood, the old mediums were vacuumed and the
cells were washed twice with PBS. PBS was removed with vacuum and 0.5 ml of
trypsin was added to each flask. Cells were incubated for 2-3 minutes at room
temperature. Detachment of cells from the flask surface was confirmed via
microscope and 5 ml of fresh RPMI 1640 (with supplements) medium was added to
deactivate trypsin. Medium was pipetted gently to scatter cells from flask surface.
Each cell line was divided into two by transferring 2.5 ml into new two 25 ml flask
for each. 2.5 ml of medium was added into each bringing the total volumes to 5 ml.
Flasks were kept in incubators at 37°C with 5% CO2.
3.2.8 Pelleting and Collecting Cells
The medium of flasks, where cells reached to optimal confluency, was aspirated and
washed with PBS twice. 0.5 ml of trypsin was used for each flask to detach cells
from the surface. Flasks were incubated at room temperature for 2-3 minutes. Fresh
medium was supplied for each and gently pipetted. Suspensions were collected to 15
ml falcon tubes and centrifuged at 1400 rpm for 5 minutes. Supernatants were
discarded and the pellets were washed with PBS twice. Tubes were centrifuged again
at 1400 rpm for 5 minutes and resulting supernatants were aspirated via vacuum
carefully. Pellets were immediately kept at -80°C.
31
3.2.9 Genomic DNA Isolation
Cell pellets were removed from -80°C refrigerator and thawed at room temperature.
Qiagen DNeasy Tissue Kit (Venlo, The Netherlands) was used for isolating genomic
DNA according to manufacturer’s recommended protocol. Nanodrop
Spectrophotometer (Nanodrop Technologies) was used for assessing the quality and
amount of genomic DNA extracted. Samples were stored at -20°C.
3.2.10 Immunohistochemical Analysis of LSAMP Protein
Immunohistochemistry (IHC) was performed on sections of 6 neuroblastoma, 1 fetal
kidney & suprarenal and 1 adult cerebellum tissues. Neuroblastoma tissues from
patients were sectioned on poly-l lysine coated slides and were provided by Dr.
Aylin Okçu Heper from School of Medicine, Department of Pathology, Ankara
University. Dr. Emin Öztaş from Gülhane Military Medical Academy (GMMA)
helped us with the IHC protocol. In brief, tissue sections were deparaffinized at 70°C
and then in Xylene. After rehydration in graded alcohol series, glass slides were
immerged in 10 mM citrate buffer, pH 6.0 and transferred into microwave for 20
minutes for antigen retrieval. Endogenous peroxidase was blocked by incubation of
slides in 0.3 % H2O2 for 30 minutes. Phosphate buffered saline (PBS) was used in all
washing steps. Tissue sections were incubated with LSAMP polyclonal antibody
used at 1:500 dilutions in blocking solution, 50 µl was used for each slide. Slides
were left overnight in humid chamber at 4 °C. Next day slides are washed with PBS
and then universal staining kit (LabVision) was used according to manufacturer
recommendations. Diaminobenzidine (DAB) was used as chromogen, and the slides
were counterstained using Mayer’s haematoxylin. Normal serum or phosphate
buffered saline were used as negative controls, instead of the primary antibodies.
Both positive and negative controls were processed in the same slides which have
two distinct regions of tissue sections. Regions close to the slide ID was used as
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positive where regions at the terminal ends were used as negative. Dark brown
staining in sections was taken as positive reaction.
3.2.11 DNA extraction from Paraffin Embedded Tissues
5 µm sections of 12 paraffin embedded tissue samples collected from neuroblastoma
patients were provided by Dr. Aylin Okçu Heper from Ankara University, Medical
School, Department of Pathology. The same patients studied for LSAMP expression
by using IHC were chosen. Boiling method protocol of Cao et al (2003) was used for
DNA extraction from paraffin embedded tissues (Cao et al., 2003). Briefly, 1 ml of
Xylene was added into the eppendorfs containing paraffin sections. Tubes were
inverted several times and incubated a few minutes at room temperature. The
samples were centrifuged for 5 min at 13,000 rpm to pellet the tissue. After obtaining
pellets, the two steps, adding Xylene and centrifugation, were repeated. The pellets
were washed by adding 100% ethanol, the tubes were inverted several times and
centrifuged as 13,000 rpm for 5 min. The supernatant was removed and the step was
repeated. 1 ml of 10 mM TE buffer was added into each tube and inverted several
times before centrifuging at 13,000 rpm for 5 min. Supernatants were removed.
100 µl of 10 mM TE/1% Tween 20 containing 200 µg proteinase K was added into
each tube and left to incubation at 55°C overnight. Next day, the tubes were heated at
97°C for 10 min to inactivate proteinase K and centrifuged at 13,000 rpm for 5 min.
Supernatants were removed into new tubes and stored at - 20 °C.
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4 RESULTS:
4.1 Genetic Analysis of LSAMP Gene Region in Tumors and Cell
Lines:
Neuroblastoma genomic DNA was isolated from paraffin sections from 6 patients (2
sections for each) and 2 neuroblastoma cell lines; SK-NA-S and CLB-MA1, were
analyzed for LSAMP. Samples 13598-1, 13598-2, 3755-1, 3755-2 and 3978-2 did not
result in amplifications for both genes (data not shown). GAPDH was used as
reference gene and GAPDH primers were used in combination with LSAMP primers
in Multiplex-PCR reactions. 2 HCC (hepatocellular carcinoma) samples, SKHep-1
and Hep-3B, which have normal copy numbers of LSAMP, were used as positive
controls. Products were loaded together with Gene Ruler DNA Ladder mix (100-
10,000 bp) in 2% agarose gels under constant voltage of 100V about 30 min. The
expected product sizes for amplified LSAMP and GAPDH genomic regions were
98bp and 143bp, respectively. Visualization with Bio-Rad Transilluminator, revealed
the banding patterns corresponding to two genomic regions. Although, GAPDH was
longer in base pairs, it was amplified in all samples, but LSAMP having smaller base
pairs, which would be easier to amplify, was not detected to be fully amplified in a
few samples. This suggested possible deletions in LSAMP genomic region in these
samples. Thus, among 6 clinical patient samples, one possible homozygous deletion
and one LOH in LSAMP region was identified.
4.1.1 Homozygous Deletion of LSAMP Gene:
Although amplification patterns of GAPDH region were similar for all patient
samples, cell lines and controls, in patient #28231 any band corresponding to LSAMP
gene could not detected which suggests a homozygous deletion of LSAMP genomic
region (Fig.6).
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Figure 6: Homozygous deletion of LSAMP region in patient #28231 is shown. Multiplex-PCR result of LSAMP and GAPDH in neuroblastoma tumor and cell lines where HCC samples SK-Hep-1 and Hep-3B were used as positive control.
4.1.2 LOH at LSAMP Genomic Region:
Although, there was a band corresponding to LSAMP genomic region in tumor
sample of patient #12116, the intensity of the band was weaker compared to GAPDH
band. This result may suggest a possible LOH in correspondent LSAMP
chromosomal region 3q13.31 (Fig.7).
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Figure 7: Possible LOH in LSAMP locus of patient sample #12116 is shown. Multiplex-PCR result of LSAMP and GAPDH in neuroblastoma tumor and cell lines where HCC sample SK-Hep-1 was used as positive control.
4.2 Assessment of LSAMP Protein in Neuroblastoma Tissues:
6 neuroblastoma tissues sectioned from the same patients analyzed for any genomic
aberrations in LSAMP gene were further assessed at the protein level. IHC was used
to detect LSAMP protein in neuroblastoma tissues. Additionally, healthy adult
cerebellum and fetal kidney & suprarenal tissues were used as positive controls.
Pictures of successful staining were shot with Zeiss Axiocam Imager A1 microscope
(Gottingen, Germany). We observed a common weak staining pattern for all tumor
samples compared to positive controls. Moreover, consistent to the result of genomic
analysis, we did not see any protein in patient sample #28231 (Fig.8).
36
37
Figure 8: LSAMP protein levels in neuroblastoma tissues revealed by IHC (A, B, C, D, E, and F). Healthy adult cerebellum and fetal kidney &suprarenal tissue sections are used as controls (G & H).
4.3 Analysis of LSAMP Expression in Brain Tumors:
In addition to genomic and protein level analyses, we also assessed the expression of
LSAMP in mRNA level in other tumors derived from neural tissues. A set of brain
tumor patient RNAs were first reverse transcribed into cDNAs and tested for
GAPDH gene levels which was used as reference gene in semi-quantitative PCR
experiments (Fig.9 & Fig 10). All samples were used in analysis of LSAMP
expression with semi quantitative PCR and Q-RT-PCR methods.
4.3.1 Semi Quantitative PCR Results:
All brain tumor samples were first tested by using primer pairs designed for
amplifying a large region involving 6 exons in LSAMP gene. Glioblastoma samples
were successfully amplified (Fig.10). Meningioma, oligodendroglioma, astrocytoma,
ependymoma and pilocytic astrocytoma samples were further analyzed in nested
PCR reactions by using primer pairs designed for amplifying a smaller region inside
the first amplification (Fig .11).
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Figure 9: LSAMP and GAPDH expression levels of the same glioblastoma samples.
LSAMP
GAPDH
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Figure 10: LSAMP expression in brain tumors; M series: Meningioma, O series: Oligodendroglioma, N series: Normal brain, P 12: Pilocytic Astrocytoma A series: Astrocytoma Grade 4, E9: Ependymoma.
Results showed us in some tumors although GAPDH is well expressed, LSAMP
expression was lower compared to normal brain samples. A table summarizing these
results showed us there is particularly a difference of expression in astrocytoma
grade 4, meningioma and glioblastomas (Table 12). In this table, weak intensity
bands are represented by “+”, medium intensity by “++” and high intensity by “+++”
signs. Although, semi quantitatively we observe the decrease in LSAMP expression,
a more precise method Q-RT-PCR was performed in next step for detecting actual
expression levels.
4.3.2 Q-RT-PCR Detection of LSAMP Expression in Brain Tumors:
We performed Q-RT-PCR experiments by using commercially obtained LSAMP
primers amplifying a small region between 100-140bp and Βeta-actin primers to use
Beta-actin expression as control. BioRad SyBr Green Kit was used to prepare master
LSAMP
GAPDH
40
mix. The results were analyzed by using delta - delta Ct method and indicate a clear
decrease in LSAMP expression in majority of meningiomas, astrocytoma grade 4 and
glioblastoma tumors compared to normal brain tissue (Table. 13, Fig.11).
The calculated fold changes suggesting LSAMP has generally lower values for
tumors compared to normal seem statistically significant at 95% confidence interval
(p-value=0.002).
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Type and Grade Brain Tumor LSAMP GAPDH Type and Grade Brain Tumor LSAMP GAPDH